Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same

ABSTRACT

The invention provides an alginate oligosaccharide and its derivatives with the degree of polymerization ranging from 2 to 22. The alginate oligosaccharide is composed of β-D-mannuronic acid linked by 1,4 glycosidic bonds. The derivatives with the reduced terminal in position 1 of carboxyl radical can be prepared by oxidative degradation. The invention also provides a process for preparing the alginate oligosaccharide and its derivatives, which includes the procedure that an alginate solution is reacted for 2 to 6 h in an autoclave at pH 2˜6 and the temperature of 100˜120° C., and adjusted pH to 7 after the reaction is stopped, after which the resultant oligosaccharide is oxidized in the presence of an oxidant to obtain an oxidative product. The alginate oligosaccharide and its derivatives of the invention can be used in the manufacture of a medicament for the prophylaxis and treatment of AD and diabetes.

TECHNICAL FIELD

The invention relates to an alginate oligosaccharide and itsderivatives, the preparation thereof, and uses of the same for treatingAlzheimer's disease (AD) and diabetes.

BACKGROUND ART

Alzheimer's disease (AD) and diabetes are currently common andfrequently-occurring disease. Especially, their incidence is increasingas the old people growing. So the prevention and cure of these diseasesare urgent problems to be solved today.

Current drugs therapy for AD are unlikely to revolutionize the treatmentof AD due to their limitation of the mere symptomatic relief or severeadverse effects. The current drugs used for diabetes mainly are insulinand other orally hypoglycemic drugs. The inconveniency for long-term useand toxicity limited their wide applications and there are actually noeffective drugs for type 2 diabetes. The recent study revealed thatamyloid-beta (A β) and amylin (IAAP) are the basic molecules of AD andtype 2 diabetes. The fibrillogenesis and subsequently increased freeoxidative radicals are the triggers of these diseases, which gives riseto the fact that the fibril formation inhibitor of amyloid-beta andamylin becomes the perspective for the cure of these diseases.

Alginates are a family of linear polysaccharide produced by brownseaweeds and some bacterial species belonging to the genera Pseudomonasand Azotobacter. These polymers are composed of two hexuronic acids,β-D-mannuronic acid (ManA) and α-L-guluronic acid (GulA), linked by 1-4bonds. Alginate belongs to high polymers with molecular weight ofseveral 10⁴ to 10⁶ with very abundant source. The polymer alginate hasbeen used for a variety of industrial purposes, e.g. as a stabilizing,thickening and gelling agent in food production and pharmaceuticalapplications. Recent developments revealed that alginate is an importantbiological active compound. Its application as a drug is largely limitedby its huge molecular weight. So the oligosaccharide degraded fromalginate by different methods is very valuable for glycochemistry,glycobiology, glycoengineering and saccharide-based drugs. Thecurrently-used methods to degrade alginate include enzymatic, physicaland chemical treatments, yet the finding of specific lyases has limitedthe application by enzymatic method. Physical method, which is usuallyused in combination with other method, couldn't get oligosaccharide withthe minimal molecular weight of 50,000. The chemical degraded methodused for polysaccharides include acidic hydrolysis method and hydrogenperoxide degraded method. The former method is limited by its capacityto get oligosaccharides with weight less than 4000 when conducted innormal temperature and pressure.

Contents of the Invention

Through deep studies by the inventors, it is found that an alginateoligosaccharide with molecular weight below 4000 can be obtained by acidhydrolysis at high temperature and pressure, and its derivatives whosereduced terminal in position 1 is carboxyl radical can be prepared inthe presence of oxidants. The invention is completed on the above basis.

The invention provides an alginate oligosaccharide and its derivativeswith low molecular weight, or pharmaceutically-acceptable salts thereof,and provides the process for preparing the same. The invention alsoprovides a medicament for the prophylaxis and treatment of AD anddiabetes with the above-mentioned low molecular alginate oligosaccharideor its derivatives, or pharmaceutically-acceptable salts thereof.

The invention relates to an alginate oligosaccharide and its derivativesrepresented by formula (I) or pharmaceutically-acceptable salts thereof,the above-mentioned oligosaccharide is composed of β-D-mannuronic acidlinked by 1,4 glycosidic bonds,

wherein, n represents an integer of 0 or any number ranging from 1 to19.

In the present invention, an example of the above alginateoligosaccharide derivatives is a compound represented by formula (II),in which the reduced terminal in position 1 is carboxyl radical,

wherein, n represents a integer of 0 or 1 to 19.

In above formula (I) and (II), preferable n is 2 to 10, and morepreferable n is 4 to 8. The reason why better biological effects oftetrasaccharide over dodecasaccharide (preferably, hexasaccharide todecasaccharide) remains unclear, which may be caused by its liability tobe recognized and accepted by cells.

The alginate oligosaccharide derivatives also include, for example, thederivatives, of which part of the hydroxyl groups in mannuronic acid aresulfated.

For example, the pharmaceutically-acceptable salts of the above alginateoligosaccharide and its derivatives can be salts of sodium, potassium,calcium, magnesium salts and the like. The sodium salts are preferred.The pharmaceutically-acceptable salts can be prepared by theconventional methods.

The invention also relates to a process for preparing above alginateoligosaccharide and its derivatives, characterized by the fact that analginate solution is reacted for about 2 to 6 h in an autoclave at pH2˜6 and the temperature of about 100˜120° C.; after which the value ofpH is adjusted to about 7. The oxidative product is obtained by theaddition of an oxidant to the alginate oligosaccharide solution.

In a preferred embodiment of the invention, a 0.5˜5% sodium alginatesolution is heated to 110° C. for 4 h in an autoclave at pH 4. After thereaction is accomplished, the reactant is sucked out and cooled, then pHis adjusted to 7 by adding NaOH solution. With stirring, the filtrate isslowly poured in to industrial alcohol which is 4 times as the volume ofthe filtrate, and stayed overnight to allow precipitation. The alcoholprecipitate is filtered off with suction to dryness, and is dehydratedby washing with absolute ethanol. A white filter cake is obtained anddried in an oven at 60° C. to give a crude alginate oligosaccharide. Thecrude alginate oligosaccharide is formulated to a 10% solution, and isprecipitated with 95% ethanol solution. The precipitate washed withabsolute ethanol, dried and formulated to a 5% solution. The solution isfiltered through a 3 μm membrane to remove impurity, then desalted on aBio-Gel-P6 column (1.6×180 cm) with 0.2 mol/L NH₄HCO₃ as mobile phaseand then the product is obtained collected. The elute is detected by thesulfate-carbazole method, collected components including sugar,concentrated under reduced pressure and desalted, and lyophilized togive the alginate oligosaccharide.

The preparations of the derivatives represented by formula (II) is areas follows: an oxidant is added and reacted for 15 min to 2 h at thetemperature of 100˜120° C. after the step when the above alginatesolution reacting for about 2 to 6 h in an autoclave at pH 2˜6 and thetemperature of about 100˜120° C. In an embodiment of the invention, 25ml of 5% copper sulfate is added to 50 ml of 10% NaOH (aq), mixedimmediately, and immediately added 40 ml of 5% alginate oligosaccharidesolution. The resultant mixture is heated in a boiling water bath untilno more brick red precipitate is generated. The mixture is centrifugedto remove precipitate. Some supernatant is taken out and added to 10%NaOH (aq) and 5% copper sulfate according to the above ratio to checkany generation of brick red precipitate. If negative, the supernatant isadded to industrial alcohol which is 4 times the volume to of thesupernatant, and stayed overnight to allow precipitation. Theprecipitate is filtered off with suction to dryness, dehydrated withabsolute ethanol repeatedly and dried in an oven at 60° C. Theseparation is preceded according to the same separation method of thealginate oligosaccharide of formula (I).

The invention also provides a pharmaceutical composition containing aneffective amount of such alginate oligosaccharide or its derivatives, orpharmaceutically-acceptable salts thereof andpharmaceutically-acceptable carriers.

The pharmaceutical composition can be a medicament for the prophylaxisand treatment of AD.

Furthermore, the pharmaceutical composition can be an amyloid-β proteinfibrils forming inhibitor and fibrils disaggregating promoter.

The pharmaceutical composition also can be a medicament for theprophylaxis and treatment of diabetes.

Moreover, the pharmaceutical composition can be used as pancreatic isletamyloid protein fibrils forming inhibitor and islet amyloid polypeptideinhibitor. In view of the current difficulty of lacking effectivemedicines for the prophylaxis and treatment of AD and diabetes, it isespecially important that the alginate oligosaccharide of the inventionis used in the manufacture of a medicament for the prophylaxis andtreatment of AD and diabetes.

DESCRIPTION OF FIGURES

FIG. 1 shows the eluting curve of the alginate oligosaccharide accordingto the invention separated by a Bio-Gel-P6 column after acid hydrolysis.

FIG. 2 shows the MALDI-TOF spectrum of the alginate oligosaccharideaccording to the invention.

FIG. 3 shows the eluting curve of the oxidative product of the alginateoligosaccharide separated by a Bio-Gel-P6 column.

FIG. 4 shows the MALDI-TOF spectrum of the oxidative product of thealginate oligosaccharide (Posotive mode).

FIG. 5 shows the effect of the alginate oligosaccharide on the latencyof AD mice induced by Aβ₁₋₄₀ tested with Morris water maze.

FIG. 6 shows the effect of the alginate oligosaccharide on the errornumbers of AD mice induced by Aβ₁₋₄₀ tested with Morris water maze.

FIG. 7 shows the protective effects of the alginate oligosaccharide onSH—SY5Y cells impaired by Aβ₂₅₋₃₅.

FIG. 8 shows the protective effects of the alginate oligosaccharide onSH—SY5Y cells impaired by Aβ₁₋₄₀.

FIG. 9 shows the inhibitory effects of the alginate oligosaccharide onnormal and heparin-induced fibril formation of Aβ₁₋₄₀.

FIG. 10 shows the destabilized effects of the alginate oligosaccharideon fibril Aβ₁₋₄₀.

FIG. 11 shows the effects of the alginate oligosaccharide on theconformation of soluted 250 μg/ml Aβ₁₋₄₀.

FIG. 12 shows the protective effects of the alginate oligosaccharide onNIT cells impaired by IAAP.

FIG. 13 shows the effect of the mixture of oxidative product of thealginate oligosaccharide on the latency of AD mice induced by Aβ₁₋₄₀tested with Morris water maze.

FIG. 14 shows the effect of the mixture of oxidative product of thealginate oligosaccharide on the swimming distance of AD mice induced byAβ₁₋₄₀ tested with Morris water maze.

FIG. 15 shows the effect of the mixture of oxidative product of thealginate oligosaccharide on the first time arriving the original plateof AD mice induced by Aβ₁₋₄₀ tested with Morris water maze.

FIG. 16 shows the effect of the mixture of oxidative product of thealginate oligosaccharide on the numbers crossing the original plate ofAD mice induced by Aβ₁₋₄₀ tested with Morris water maze.

FIG. 17 shows the protective effects of the mixture of oxidative productof the alginate oligosaccharide on NIT cells impaired by IAAP.

EMBODIMENTS 1 Preparation of the Alginate Oligosaccharide

One gram of sodium polymannanuronate (average molecular weight of 8,235Da, provided by Lantai Pharm. LTD., Ocean University of China) is addedto distilled water to obtain a 1% solution, adjusted pH to 4 with HCl,placed into an autoclave and heated at 110° C. for 4 h. After cooling,the solution of pH is adjusted to 7 with NaOH (aq.). With stirring, thefiltrate is slowly poured into industrial alcohol which is 4 times thevolume of the filtrate, and stayed overnight to precipitate. The alcoholprecipitate is filtered off with suction to dryness, and is dehydratedby washing with absolute ethanol. A white filter cake is obtained anddried in an oven at 60° C. to give a crude alginate oligosaccharide.

The crude alginate oligosaccharide is formulated to a 10% solution, andis precipitated with 95% ethanol solution. The precipitate is washedwith absolute ethanol, after drying, formulated to a 5% solution. Thesolution is filter through a 3 μm membrane to remove impurity, thendesalted on a Bio-Gel-P6 column (1.6×180 cm) with 0.2 mol/L NH₄HCO₃ asmobile phase and collected. The elute is detected by thesulfate-carbazole method, collected components including sugar,concentrated under reduced pressure and desalted on G-10 column. Theouter volume component is further separated by a Bio-Gel-P10 column andlyophilized to give a series of alginate oligosaccharide (FIG. 1).

2 Preparation of the Oxidative Product of the Alginate Oligosaccharide

Five gram of above prepared alginate oligosaccharide is formulated to a5% solution. 25 ml of 5% copper sulfate is added to 50 ml of 10% NaOH(aq), and mixed immediately, then add 40 ml solution of 5% alginateoligosaccharide. The resultant mixture is heated in a boiling water bathuntil no more brick red precipitate is generated. The mixture iscentrifuged to remove precipitate. A few of supernatant is taken out andadded to 10% NaOH (aq) and 5% copper sulfate according to the aboveratio to check for brick red precipitate. If negative, the supernatantis added to industrial alcohol which is 4 times the volume of thesupernatant, and stayed overnight to allow precipitation. Theprecipitate is filtered off with suction to dryness, dehydrated withabsolute ethanol repeatedly and dried in an oven at 60° C. Thus a crudeoxidative product of alginate oligosaccharide is obtained.

The crude oxidative product of alginate oligosaccharide is formulated toa 10% solution, and is precipitated with 95% ethanol solution. Theprecipitate is washed with absolute ethanol, after drying, formulated toa 5% solution. The solution is filter through a 3 μm membrane to removeimpurity, then desalted on a Bio-Gel-P6 column (1.6×180 cm) with 0.2mol/L NH₄HCO₃ as mobile phase and collected. The elute is detected bythe sulfate-carbazole method, collected components including sugar,concentrated under reduced pressure and desalted on G-10 column. Theouter volume component is further separated by a Bio-Gel-P10 column andlyophilized to give a series of oxidative product (FIG. 2).

3 Structure Identification of the Alginate Oligosaccharide

The structure of oligosaccharide fraction obtained from the preparationof the alginate oligosaccharide is identified. The oligosaccharide isconfirmed to be an alginate oligosaccharide composed of β-D-mannuronicacid linked by 1,4 glycosidic bonds. The structural formula is:

wherein, n represents an integer of 0 or any from 1 to 19.

The fraction at about 292 ml of the elute (the fraction labeled as “6”in FIG. 1, hereinafter referred to as Component 6) is taken as anexample to illustrate the structure analysis of above oligosaccharide.

3.1 Ultraviolet Absorption Spectrogram

The oligosaccharide fraction at about 292 ml of the elute is diluted toan appropriate concentration, and scanned at 190 nm˜400 nm with UV-2102UV-VIS spectrophotometer. It is found that no specific absorption peakappeared in ultraviolet region, indicating that the structure is void ofconjugated double bond. However, non-specific absorption peak appearedat 190˜200 nm Thus, during desalting the oligosaccharide, it can beon-line detected at above ultraviolet region.

3.2 Infrared Spectroscopy Analysis

Five hundred microgram of above oligosaccharide fraction is weighed.Infrared spectroscopy is determined with NEXUS-470 intelligent infraredspectrometer by KBr pellet. The peaks at 3420.79, 3214.64, and 2924.61cm⁻¹ are attributable to symmetry stretching vibrations of hydroxylgroup; the peak at 1600.25 cm⁻¹ is attributable to symmetry stretchingvibration of carbonyl of carboxylate; the peak at 1406.54 cm⁻¹ isattributable to shearing vibration of hydroxyl group; the peak at1146.42 cm⁻¹ is attributable to symmetry stretching vibration of C—Obond of carboxyl; the peak at 1045.77 cm⁻¹ is attributable toanti-symmetry stretching vibrations of anhydro ether; and the peak at804.02 cm⁻¹ is attributable to anti-symmetry stretching vibrations ofmannuronic acid cyclic skeleton. It is demonstrated that such compoundhas carboxyl, hydroxyl and mannuronic acid cyclic skeleton.

3.3 MS Analysis

MS analysis is performed with BIFLEX II type MALDI-TOF mass spectrometer(Bruker Daltonics Co.). As seen from FIG. 2, the peak of mz 1073.9 isthe molecular ion peak [M-H]⁻¹; mz 1096.6 is [M+Na-2H]⁻¹; mz 1028.0 is[M-H2O—CO—H]⁻¹; mz 821.2 is [M-ManA-CH2O-2H2O—H]⁻¹; mz 704.3 is[M-2ManA-H2O—H]⁻¹; mz 634.4 is [M-2ManA-2(CH2O)—CO—H]⁻¹; mz 536.5 is[M-2H]²⁻; and mz 357.4 is [M-3H]³⁻. In ESI-MS spectrum of aboveoligosaccharide fraction, the molecular ion peak is mz 1073.9,indicating that molecular weight is 1074.

TABLE 1 MS analysis of the alginate oligosaccharide (Component 6)Fragment ions m/z [M − H]⁻¹ 1073.9 [M + Na—2H]⁻¹ 1096.6 [M − H₂O—CO—H]⁻¹1028.0 [M-ManA-CH₂O—2H₂O—H]⁻¹ 821.1 [M-2ManA-H₂O—H]⁻¹ 704.3[M-2ManA-2(CH₂O)—CO—H]⁻¹ 634.4 [M − 2H]²⁻ 536.5 [M − 3H]³⁻ 357.4

3.4 Nuclear Magnetic Resonance Spectroscopy of the AlginateOligosaccharide

¹H NMR and ¹³C NMR of the alginate oligosaccharide represented byformula (I) (n=4) are obtained by JNM-ECP600 NMR spectrometer. Theresults are shown in table 2 and 3.

TABLE 2 ¹H-NMR of the alginate oligosaccharide (Component 6) Chemicalshift (ppm) H-1 H-2 H-3 H-4 H-5 r α 5.21 3.98 4.03 4.04 4.16 r β 4.913.99 3.77 3.90 3.77 m α 4.69 4.03 3.75 3.93 3.69 m β 4.64 4.03 3.75 3.653.69 n 4.63 3.74 3.63 3.75 4.01

TABLE 3 ¹³C-NMR of the alginate oligosaccharide (Component 6) Chemicalshift (ppm) C-1 C-2 C-3 C-4 C-5 C-6 r α 93.54 70.06 69.02 78.37 72.60175.84 r β 93.74 70.42 71.60 78.28 76.08 175.84 m 99.08 70.63 71.4378.07 75.90 175.41 n 100.15 68.48 72.47 76.27 70.05 175.27

According to above analysis results, it is confirmed that the alginateoligosaccharide in above fraction is mannuronic hexasaccharide with thefollowing structure (Ia):

3.5 Determination of the Content of Mannuronic Acid in the AlginateOligosaccharide (¹H-NMR Spectroscopy)

Constitute of the alginate oligosaccharide is determined by highresolution ¹H-NMR to quantify the ratio of mannuronic acid to guluronicacid (MG) in the alginate oligosaccharide according to the signalintensity of proton of anomeric carbon. Three to five mg of dried sampleis weighed and dissolved in D₂O at neutral pD. Three hundred microgramof EDTA is added. The sample is determined by Bruker DPX-300 NMRspectrometer. The spectrum is reported at 70° C., making the peak of D₂Ofar away from anomeric proton resonance region. The signal relativeintensity is expressed by the integral of the peak area. The resultsshown that H-1 signals of M radical appeared at 4.64 ppm and 4.66 ppm(i.e. H-1 signals of M radical in MM and MG sequence respectively); allof H-1 signals of G radical appeared at 5.05 ppm (double peak). Therelative content of M and G in the sample can be expressed by their H-1peak intensity, as the following equation:

${M\mspace{14mu} \%} = {\frac{I_{4.64} + I_{4.66}}{I_{4.64} + I_{4.66} + I_{5.05}} \times 100\%}$

wherein, I represents the peak intensity, expressed by the integral ofthe peak area.

The relative content of D-mannuronic acid in the sample is 98.07% byabove method, indicating that the alginate oligosaccharide is mainlycomposed of mannuronic acid.

4 Structure Identification of the Oxidative Product of the AlginateOligosaccharide

The structure of the oligosaccharide oxidative product fraction obtainedfrom the preparation of the oxidative product of the alginateoligosaccharide is identified. The oxidative product is confirmed to bea derivative of the alginate oligosaccharide composed of β-D-mannuronicacid linked by 1,4 glycosidic bonds, in which the reduced terminal inposition 1 is carboxyl radical. The structural formula is:

wherein, n represents a integer of 0 or 1 to 19.

Component 6 is taken as an example to illustrate the structure analysisof above oligosaccharide oxidative product.

4.1 Ultraviolet Absorption Spectrogram

An appropriate oxidative product is diluted to a certain concentrationwith distilled water, and scanned with Shimdzu UV-260 UVspectrophotometer (190 nm˜700 nm) at full wavelength. It is found thatno specific absorption peak appeared in ultraviolet and visible lightregion.

4.2 Infrared Spectroscopy Analysis

Infrared spectrum of the oxidative product of the alginateoligosaccharide is determined by NICOLE NEXUS-470 intelligent infraredspectrometer. The results are shown in table 4.

TABLE 4 IR spectrum of the oxidative product of the alginateoligosaccharide Absorption peak (cm⁻¹) Type of vibration Group Intensity3400.56 υ_(OH) —OH s 3219.02 υ_(OH) —OH s υ_(CH) —COOH 2924.65 υ_(OH)—COOH m 1599.76 υ_(C═O) —COOH s 1405.95 υ_(C—O) —COOH s 1296.26 δ_(O—H)—OH m 1037.84 υ_(as)(C—O—C) anhydro ether m 817.14 υ_(as) (sugar ring)mannuronic acid cyclic m skeleton 669.80 γ_(OH) —OH m

4.3 ¹H-NMR Analysis

¹H-NMR and ¹³C-NMR of the oxidative product are obtained by BrukerAuance DPX-300 NMR spectrometer. As seen from ¹H-NMR spectrum, it ismainly composed of the signals of six hydrogen atoms in β-D-mannuronicacid. After coupling pattern of each signal is assigned, it is foundthat the oxidative product of the alginate oligosaccharide is mainlycomposed of mannuronic acid. If the reduced terminal in position 1 isaldehyde group, chemical shifts of H-1 α and β should be 5.11 ppm and4.81 ppm, respectively. Since the reduced terminal in position 1 of thealginate oligosaccharide is oxidated to carboxyl from aldehyde group,H-1 disappeared, thus signals at 5.11 ppm and 4.81 ppm disappeared. Asseen from ¹³C-NMR spectrum, it is mainly composed of the signals of sixcarbon atoms in β-D-mannuronic acid. After coupling pattern of eachsignal is assigned, it is found that intermediate molecule is mainlycomposed of mannuronic acid. Compared with the spectrum of intermediate,the signal of the reduced terminal C-1 of mannuronic acid (94 ppm)disappears. The signal of the reduced terminal C-1 (175.81 ppm) isshifted towards low field. The reason is that the reduced terminal inposition 1 of the alginate oligosaccharide is oxidated to carboxyl fromaldehyde group, thus chemical shift of C-1 is changed from 94 ppm ofaldehyde group to 175.81 ppm of carboxyl.

4.4 MS Analysis

MS analysis is performed with BIFLEX III type MALDI-TOF massspectrometer (Bruker Daltonics Co.). The results are shown in FIG. 4. Asseen from FIG. 4, the peak of mz 1113.7 is [M+Na]⁺¹; mz 1113.7 is[M−O+Na]⁺¹; mz 1083.7 is [M-CH₂O+Na]⁺¹; mz 1067.6 is [M-CH₂O—O+Na]^(+l);mz 1053.6 is [M-2(CH₂O)+Na]⁺¹; mz 979.6 is [M-3(CH₂O)—CO₂+Na]⁺¹; mz921.6 is [M-4(CH₂O)—CO₂—CO+Na]⁺¹. MS analysis of the oxidative productof the alginate oligosaccharide is shown in table 5.

TABLE 5 MS analysis of the oxidative product of the alginateoligosaccharide Fragment ions m/z [M + Na]⁺¹ 1113.7 [M-O + Na]⁺¹ 1097.7[M-CH₂O + Na]⁺¹ 1083.7 [M-O—CH₂O + Na]⁺¹ 1067.6 [M-2 (CH₂O) + Na]⁺¹1053.6 [M-3 (CH₂O)—CO₂ + Na]⁺¹ 979.6 [M-4 (CH₂O)—CO₂—CO + Na]⁺¹ 921.6

In MALDI-TOF spectrum of the oxidative product of the alginateoligosaccharide, the peak of mz 1113.7 is [M+Na]⁺¹, indicating thatmolecular weight of the oxidative product is 1090.7. As the molecularweight increased sixteen compared with that of acid hydrolyzed alginateoligosaccharide (M=1075), that is, the molecule increased an oxygenatom, it is considered that the alginate oligosaccharide is oxidatedduring the preparation.

According to above analysis results, the structure of the oxidativeproduct of the alginate oligosaccharide is the formula (IIa):

5 Evaluation of Oligomannurarates on Alzheimer's Disease (AD)

The 6-mer separated with Bio-Gel-P6 column is used as an example to showtheir activity in the following experiments.

5.1 Effects of 6-Mer on AD Mice Induced by Aβ₁₋₄₀

Male Balb/c mice (18-22 g, purchased from Laboratory Centre of ShandongUniversity) are randomly assigned to six groups as follows:6-mer-treated groups (administered orally for 7 consecutive days atdoses of 15, 30 and 60 mg/kg daily prior to Aβ₁₋₄₀ injection), HuperzineA-treated (HBY) group (administered orally with HBY at a dose of 0.2mg/kg once a day), Aβ₁₋₄₀-injected group (model group, only administeredwith saline daily), and vehicle group (control group, only administeredwith saline daily). On day 8, mice are injected with aged Aβ₁₋₄₀ exceptthe vehicle group as the method of reference (Jhoo J H at al, β-amyloid(1-42)-induced learning and memory deficits in mice: involvement ofoxidative burdens in the hippocampus and cerebral cortex. BehaviouralBrain Research (2004) 155: 185-196) to induce the AD model. Followingsurgery, mice are further treated with drugs or saline. The effects of6-mer on Aβ-induced AD model are evaluated with Morris water maze taskand step-through test. MDA, superoxide dismutase (SOD), glutathioneperoxidase (GSH-Px), Na⁺, K⁺-ATPase, AchE and CHAT activities in thebrain regions are determined using the respective kits (NanjingJiancheng Bioengineering Institute, Nanjing, PR China).

Morris water maze test is conducted in 17 days post drug administration.The results of acquisition trials showed that Aβ-treated mice displayedlonger escape latency, comparable with the controls. However, thisincreased escape latency is shortened in a dose-dependent manner by6-mer (table 6). In a spatial probe trial (table 7), mice in theAβ-treated group exhibited lower latency bias than the control mice(P<0.05), and the latency bias of mice is elevated significantly afteradministration of 6-mer or HBY, compared with that of the Aβ-treatedgroup (P<0.05). HBY, a selective acetylcholinesterase inhibitor, is lesspotent than 6-mer both in terms of acquisition and spatial probe trials.

Three days after the Morris water maze test, mice are further trainedfor step-through passive avoidance task. The results are shown in FIGS.5 and 6. Each group has 8 animals. The data are presented as mean±SE.The symbol # means significantly different compared with control group(P<0.05). * means significantly different compared with model group(P<0.05). The step-through latency is prolonged (FIG. 5), and the numberof errors is significantly reduced in 6-mer-treated group in adose-dependent fashion (FIG. 6), compared with those of theAβ₁₋₄₀-treated group, indicating that 6-mer markedly anddose-dependently improves Aβ₁₋₄₀-induced amnesia. Moreover, HBY is alsoless potent than 6-mer, both in terms of learning and memory tasks.

TABLE 6 Effects of 6-mer on escape latency of AD mice induced by Aβ₁₋₄₀tested with Morris water maze ( x ± SE) Dose Escape latency (s) Group(mg/kg) n 1st day 2nd day 3rd day Control — 12 49.40 ± 8.39 54.30 ±11.39 42.80 ± 10.04 Model — 14  87.20 ± 7.58^(##) 93.46 ± 8.67^(# ) 97.31 ± 8.65^(##) 6-Mer 15 14  90.07 ± 10.71 83.29 ± 9.53  72.83 ±12.50 30 14 77.71 ± 8.69 71.69 ± 10.11 68.45 ± 14.46 60 13  56.92 ±9.92*  63.57 ± 10.54*  62.50 ± 13.10* HBY 0.2 14 76.29 ± 9.74  64.58 ±10.36*  63.83 ± 10.12* ^(#)p < 0.05, ^(##)p < 0.01 vs control; *p < 0.05vs model

TABLE 7 Effects of 6-mer on the probe trial of AD mice induced by Aβ₁₋₄₀tested with Morris water maze ( x ± SE) Group Dose (mg/kg) n Latencybias(%) Control — 12 29.48 ± 5.47 Model — 14  11.83 ± 3.33^(#) 6-Mer 1514 19.67 ± 5.15 30 14 22.99 ± 5.79 60 13  28.44 ± 6.08* HBY 0.2 14 22.18± 5.93 ^(#)P < 0.05 vs control; *P < 0.05 vs model

Following the Step-through test, rats are decapitated. Cerebral cortexand hippocampus are dissected, and stored at −80° C. until use. MDA,SOD, GSH-PX, Na⁺, K⁺-ATPase, AchE and CHAT activities in the brainregions are determined using the respective kits.

(1) Effects of 6-Mer on the ChAT Activity of AD Mice

The ChAT activity in cerebral cortex is markedly decreased aftertreatment with Aβ, as compared to control group (p<0.05). However itsactivity is increased after the treatment of 6-mer and HBY, with 30 and60 mg/kg of 6-mer and HBY have the significantly difference (table 8).

TABLE 8 Effects of 6-mer on the cerebral cortex ChAT activity ofAβ₁₋₄₀-induced AD mice (n = 10, x ± SE) Group Dose (mg/kg) ChAT activity(pmol/mg prot./min) Control — 92.17 ± 2.95 Model — 77.26 ± 4.9^(#) 6-Mer15 90.94 ± 3.77 30  99.98 ± 5.07** 60  94.69 ± 5.83* HBY 0.2  100.70 ±5.99** ^(#)P < 0.05 vs control; *P < 0.05, **P < 0.01 vs model

(2) Effects of 6-Mer on the SOD Activity of AD Mice

The SOD activity in brain is decreased after treatment with Aβ, but hasno statistically significance as compared with control group. Itsactivity is increased both in cerebral cortex and hippocampus after thetreatment of 6-mer at dosage of 60 mg/kg, indicating 6-mer has theantioxidant activity (table 9).

TABLE 9 Effects of 6-mer on the brain SOD activity of Aβ₁₋₄₀-induced ADmice (n = 10, x ± SE) SOD activity (NU/mg prot) Group Dose (mg/kg)cerebral cortex hippocampus Control — 53.48 ± 1.56 66.35 ± 4.74 Model —49.99 ± 2.41 62.24 ± 4.16 6-Mer 15 49.35 ± 2.27 69.76 ± 6.12 30 51.84 ±2.07 61.72 ± 4.27 60  57.50 ± 2.51*  79.97 ± 7.34* HBY 0.2 48.95 ± 2.1369.91 ± 6.51 *P < 0.05 vs model

(3) Effects of 6-Mer on the MDA Content of AD Mice

The MDA content in brain has no statistically significance as comparedwith control group. Its content is decreased both in cerebral cortex andhippocampus after the treatment of 6-mer at dosage of 30 and 60 mg/kg,indicating 6-mer has the ability of clearance of free radicals (table10).

TABLE 10 Effects of 6-mer on the brain MDA content of Aβ₁₋₄₀-induced ADmice (n = 10, x ± SE) MDA content (nmol/ml) Group Dose (mg/kg) cerebralcortex hippocampus Control —  2.61 ± 10.22 4.75 ± 0.66 Model — 2.18 ±0.23 5.17 ± 0.47 6-mer 15 1.79 ± 0.15 4.28 ± 0.82 30 1.87 ± 0.18  2.48 ±0.43** 60  1.47 ± 0.11**  2.18 ± 0.43** HBY 0.2  1.61 ± 0.13*  2.26 ±0.39** *P < 0.05, **P < 0.01 vs model

(4) Effects of 6-Mer on the GSH-PX Activity of AD Mice

The GSH-PX activity in brain is decreased after usage of Aβ withsignificant difference to control group in hippocampus (p<0.05). Itsactivity is increased in cerebral cortex after the treatment of 6-mer atdosage of 60 mg/kg (p<0.05, table 11).

TABLE 11 Effects of 6-mer on the brain GSH-PX activity of Aβ₁₋₄₀-inducedAD mice (n = 10, x ± SE) GSH-PX (U/mg prot) Group Dose (mg/kg) cerebralcortex hippocampus Control — 7.81 ± 1.20 5.39 ± 0.67 Model — 6.43 ± 1.56 3.13 ± 0.58^(#) 6-Mer 15 8.53 ± 0.86 4.13 ± 0.58 30 7.12 ± 1.10 4.25 ±0.54 60 10.75 ± 1.80* 4.81 ± 0.95 HBY 0.2 8.85 ± 1.33  5.29 ± 0.99*^(#)P < 0.05 vs control; *P < 0.05 vs model

(5) Effects of 6-Mer on the Na⁺, K⁺-ATPase Activity of AD Mice

The Na⁺, K⁺-ATPase activity in brain is significantly decreased aftertreatment with Aβ as compared with control group, while its activity ismarkedly increased after the treatment of 6-mer (P<0.05, table 12).

TABLE 12 Effects of 6-mer on the brain Na⁺, K⁺-ATPase activity ofAβ₁₋₄₀-induced AD mice (n = 10, x ± SE) ATPase activity (μmol Pi/mgprot./hour) Group Dose (mg/kg) cerebral cortex hippocampus Control —1.06 ± 0.05  2.65 ± 0.38 Model — 0.89 ± 0.06^(#)  1.62 ± 0.17^(#) 6-Mer15 1.08 ± 0.06* 2.10 ± 0.29 30 1.09 ± 0.08* 2.07 ± 0.23 60 1.08 ± 0.05* 2.52 ± 0.25* HBY 0.2 0.91 ± 0.05   2.35 ± 0.43 ^(#)P < 0.05 vs control;*P < 0.05 vs model

5.2 Protective Effects of 6-Mer on Neurons Impaired by Aβ in Vitro

The cerebral cortex neurons of rat are cultured as the method ofreference (Banker G A, et al, Rat hippocampal neurons in dispersed cellculture. Brain Res, 1977, 126:397-425). The cells cultured for 1 weekare used in this experiment. Briefly, primary cultured neurons culturedfor 7 days are seeded in 96-well plates at a density of 1×10⁵ cells perwell, and grown in Dulbecco's modified Eagle's medium (DMEM, Hyclone,Logan, Utah, U.S.A) containing 10% fetal bovine serum (FBS, Hyclone,Logan, Utah, U.S.A). The day after plating, cells are pretreated withvarying concentrations of 6-mer (final concentration of 0, 10, 50, 100μg/ml) for 24 h, followed by the addition of aged Aβ₂₅₋₃₅ (Firstlyresolved in distilled water with concentration of 1 mg/ml, then stayedat 37° C. for 7 days to get aged Aβ₂₅₋₃₅) with final concentration of 30μM. After 24 h at 37° C., 10 μl MTT with concentration of 5 mg/ml areadded. After 4 hour at 37° C., the supernatant are removed and 150 μlDMSO are added. Then the absorbance at 570 nm (630 nm as reference) isrecorded with an ELISA reader (Rainbow, TECAN, Austria).

The results showed that the survival of the cells are significantlyreduced after treatment with aged Aβ₂₅₋₃₅ (the survived cells reduced to54.5±8.9%, P<0.001 compared to control). 6-mer at dosage of 10, 50, 100μg/ml could increase the survived cells impaired by Aβ₂₅₋₃₅ in adose-dependent manner (the survived cells are 72.0±11.2%, 77.1±8.1% and82.3±11.6% respectively).

6-mer has the similar protective effects on neuron cell line SH—SY5Yimpaired with aged Aβ₂₅₋₃₅ and Aβ₁₋₄₀ (FIG. 7,8). 30 μM Aged Aβ₂₅₋₃₅ and2 μM Aβ₁₋₄₀ could impair the cells with the survived cells of 73.3±9.4%and 64.1±2.5% when treated for 48 h. 6-mer at dosage of 50, 100 μg/mlcould inhibit the neurotoxicity of Aβ.

The above experiments revealed that 6-mer could shorten the escapelatency and increase the numbers of crossing the original plate andshorten the time of arriving the original plate on AD mice induced withAβ₁₋₄₀, implying its behavioural improvement activity in Aβ-treatedmice. This in vivo result as well as the in vitro protective effects onprimary and neuron cell lines supported that 6-mer has the anti-ADactivity.

6 Action Mechanism Study of 6-Mer on AD 6.1 Effects of 6-Mer on theApoptosis of Cell Line SH—SY5Y Induced by Aβ₂₅₋₃₅

SH—SY5Y cells are seeded in 6-well plates at a density of 2×10⁵ cellsper well, and grown in DMEM medium containing 10% FBS. The day afterplating, cells are pretreated with varying concentrations (0, 50, 100μg/ml) of 6-mer for 24 h, followed by the addition of 30 μM agedAβ₂₅₋₃₅. After 48 h at 37° C., cells are collected, washed, and stainedwith propidium iodide (5 μg/ml containing 100 U/ml RNase). Then thecells (1×10⁴) are used for cell-cycle analysis by Flow cytometry.

The results found that SH—SY5Y cells treated with 30 μM aged Aβ₂₅₋₃₅show 24.8±1.9% hypodiploid cells. However, pretreatment with 50 and 100μg/ml 6-mer for 24 h suppressed apoptosis induced by aged Aβ₂₅₋₃₅ for 24h, and the observed percentages of hypodiploid cells are 10.2±1.3% and5.1±0.7%, respectively.

Further study revealed that 6-mer also significantly arrested apoptoticcascade by reversing overload of intracellular calcium ion and ROSaccumulation, thus up-regulating Bcl-2 and down-regulating P53 andCaspase-3 expression induced by Aβ. All these factors contribute to thetherapeutic potential of 6-mer in the treatment of AD.

6.2 Molecular Mechanism of 6-Mer on Anti-Neuron Toxicity of Aβ

(1) Effects of 6-Mer on the Fibril Formation of Aβ₁₋₄₀

The Th-T fluorometric assay is used to measure β-sheet formation of Aβfibril. To evaluate the effects of 6-mer on fibrillogenesis, 1.0 mg/mlmonomeric Aβ₁₋₄₀ is incubated alone or with 6-mer (final concentrationof 0, 10, 50, 100 μg/ml) or heparin or with the concomitant presence of6-mer and heparin at 37° C. for 24 h and 48 h in 150 mM phosphate buffer(pH 7.4). After incubation, 10 μl of above solution is added to Th-T ata final concentration of 3.0 μM in a volume of 0.3 ml of 50 mM phosphatebuffer (pH 6.0). Fluorescence is monitored at Em=450 nm and Ex=480 nm,using Jasco FP6200 fluorescence spectrophotometer. The fluorescence ofeach sample is corrected by subtracting from the background fluorescenceof 3.0 μM Th-T. Data from three identical samples in separateexperiments are averaged to provide the final values.

The results show that Th-T fluorescence intensity increased in thepresence of Aβ₁₋₄₀. However, 6-mer (at dosage of 10, 50, 100 μg/ml)decreases Th-T fluorescence intensity significantly 10.46±0.94,9.18±1.32 and 7.81±1.38 (p<0.05, 0.05 and 0.001). The same effects of6-mer on the fibril formation of Aβ₁₋₄₀ are studied with TEM(JEM-1200EX) (FIG. 9). A is the photo of Aβ₁₋₄₀ alone incubated for 24h; B is the mixture of Aβ₁₋₄₀ and heparin incubated for 24 h; C is themixture of Aβ₁₋₄₀ and 6-mer incubated for 24 h; D is the mixture ofAβ₁₋₄₀, heparin and 6-mer incubated for 24 h; F is Aβ₁₋₄₀ aloneincubated for 48 h; G is the mixture of Aβ₁₋₄₀ and 6-mer incubated for24 h; H is the mixture of Aβ₁₋₄₀, heparin and 6-mer incubated for 24 h.These findings indicate that 6-mer suppresses both fibrillogenesis ofAβ₁₋₄₀ alone and is facilitated by heparin.

(2) Effects of 6-Mer on the Destability of Aβ₁₋₄₀ Fibril.

Distilled water-resolved Aβ₁₋₄₀ is initially allowed to assemble for 1week, after which it is exposed to 6-mer or heparin for 3 days. TEMreveals that Aβ₁₋₄₀ alone leads to the formation of long, twisted fibersfollowing 7 days incubation at 37° C. (FIG. 10A). In the presence ofheparin for an additional 24 h, the long, twisted fibers become muchdenser and longer compared to those formed in the absence of heparin(FIG. 10B). Notably, in the presence of 6-mer, the long and aggregatedAβ fibers are turned into small irregular short fibers (FIG. 10C). Thesefindings suggest that 6-mer is capable of reversing preformed Aβ fibril,highlighting the destabilizing action of 6-mer on preformed Aβ fibriland thus potentially therapeutic intervention.

(3) Effects of 6-Mer on the β-Sheet Formation of Aβ₁₋₄₀

Fibrillogenesis strictly depends on the structural transition of Aβpeptide from α-helix or random coils to an organized β-sheetconformation. CD spectra (J-500A, JASCO, Japan) of monomeric Aβ₁₋₄₀ (250μg/ml in TBS (100 mM Tris, 50 mM NaCl, pH7.4)) incubated for 12 h ismainly characterized by β-sheet secondary structure (FIG. 11A). Thesimultaneous exposure of monomeric Aβ₁₋₄₀ to 6-mer (100 μg/ml) for 12 hprevents the β-sheet formation (FIG. 11C). However, heparinsignificantly accelerates the conformational transition to β-sheetstructure (FIG. 11A).

(4) Interaction Between 6-Mer and Aβ

SPR technique (BIAcore X, Uppsala, Sweden) is used to characterize theinteraction of 6-mer and Aβ. Different degree of aged Aβ₁₋₄₀ (aged for0, 0.5, 1, 2, 4, 6 days at 37° C.) in a series concentrations are flowedthrough the 6-mer-immobilized sensor chip. The flow rate is 5 μl/min,and the injection volume is 10 μl. The binding sensorgramm is recordedand the sensor chip is regenerated with 2M NaCl.

The results showed that different degrees of aged Aβ₁₋₄₀ all could bindto 6-mer. The binding affinity is weak of fresh Aβ₁₋₄₀ with K_(D) valueof 6.85 E-07 M. The binding affinity increased with aged degree (K_(D)values are 1.07 E-07, 9.06 E-08, 5.43 E-08, 2.15 E-08, 1.45 E-08 Mrespectively), almost stable after aged for 2 days.

Further studies reveal that 6-mer bind to the full length Aβ throughHis13˜Lys16, while Ser26˜Lys28 is the binding site of 6-mer on Aβ₂₅₋₃₅.The binding of 6-mer with fresh Aβ might contributed to its inhibitionon fibrillogenesis of Aβ. The binding of 6-mer with aged Aβ mightcontributes to its disassemble ability on fibril Aβ.

The above studies reveal that the molecular mechanisms are attributed tothe fact that 6-mer both hinder the whole fibrillogenetic process andparticularly disassemble the preformed Aβ fibril via binding HHQKepitopes on Aβ. These results indicate that 6-mer, acting as a full Aβcascade antagonist, is a potential preventive and therapeutic candidatefor AD, which provides the proof of principle for a new strategy forcure of AD.

7 Study of 6-Mer on Diabetes Models

7.1 Protective effects of 6-mer on pancreatic beta-cells impaired byamylin

The pancreatic beta-cells cell line NIT is cultured with DMEM containing10% FBS. The cells are planted in 96-well plate in density of 1×10⁴cells/well. The day after plating, cells are pretreated with varyingconcentrations of 6-mer (final concentration of 0, 10, 50, 100 μg/ml)for 24 h, followed by the addition of aged amylin with finalconcentration of 30 μM. After 48 h at 37° C., 10 μl MTT withconcentration of 5 mg/ml are added. After 4 hour at 37° C., thesupernatants are removed and 150 μl DMSO are added. Then the absorbanceat 570 nm (630 nm as reference) is recorded with an ELISA reader.

The results show that 6-mer could increase the survived cells impairedby amylin in a dose-dependent manner (FIG. 12). The data implies that6-mer has the protective effects on cells impaired with amylin. Therepeated wells are 6. The data is shown as mean±SE. ## meanssignificantly different compared with control (p<0.01). * and ** meanssignificantly different compared with model (p<0.05 and p<0.01).

7.2 Effects of 6-Mer on the Diabetic Mice Induced by Streptozotocin(STZ)

Sixty male NIH mice (weighed 18-22 g) are randomly divided to control,model, 50, 150, 450 mg/kg 6-mer and 5 mg/kg dimethyldiguanide groups.The mice are injected intraperitoneally with 150 mg/kg STZ exceptcontrol group at the 1st day. Then the mice are given accordingly drugsconsecutively for 10 days. The blood was taken to detect the glucoseconcentration after 30 min of final drug administration. The resultsshow that the blood glucose concentration is increased after treatmentwith STZ. The concentration are lowed when treated with 6-mer. This dataindicated that 6-mer have therapeutic activity on STZ-induced diabeticmice (table 13).

TABLE 13 Effects of 6-mer on the blood glucose concentration of diabeticmice induced by STZ ( x ± SD) Blood glucose concentration Group Dose(mg/kg) Number (mg/dL) Control — 10 150.6 ± 36.8  Model — 10  312.4 ±89.2^(###) 6-mer 50 10 219.4 ± 67.8*  150 10 179.6 ± 69.8** 450 10 162.5± 3**   Dimethyldiguanide 5 10 201.6 ± 58.9** ^(###)P < 0.05 vs control;*P < 0.05, **p < 0.01 vs model

The same experiments are conducted with 2-mer to 22-mer alone or theirmixture or oxidative products. The results are similar to that of 6-merto indicate their potential activity on AD and diabetes. FIGS. 13-16showed the behavioral results of mixture of oxidative products ofalginate on AD mice induced by Aβ₁₋₄₀ injected to brain. Each group has8 animals. The data presented as mean±SE. The symbol # and ## stand forsignificantly different compared to control group (p<0.05, p<0.01), and*and ** stand for significantly different compared to model group(p<0.05, p<0.01). This data revealed that oligomannurarate could improvethe learning and memory ability of AD mice. FIG. 17 showed theprotective results of mixture of oxidative products of alginate on celllines NIT impaired by amylin. Each group has 6 wells. The data presentedas mean±SD. The symbol ## stands for significantly different compared tocontrol group (p<0.01), and * and ** stand for significantly differentcompared to model group (p<0.05, p<0.01). This data revealed thatoligomannurarate has potential activity on diabetes.

8 Statistic Analysis

Data are expressed as mean±standard deviation (SD) or standard error(SE), as indicated. Student t-test and analysis of variance (ANOVA)followed by Newman-Keuls post hoc test are performed to assess thedifferences between the relevant control and each experimental group.Results of water maze training are specifically assessed by mixed designANOVA. P-values of <0.05 and 0.01 are regarded as statisticallysignificant.

Based on the above results, the pharmaceutical composition containing aneffective amount of the mannuronic acid oligosaccharide derivatives andpharmaceutically-acceptable carriers can be easily prepared. The saidpharmaceutical composition is any one of a medicament for theprophylaxis and treatment of AD, an amyloid-β protein fibrils forminginhibitor, a medicament for the prophylaxis and treatment of diabetes,an islet amyloid protein fibrils forming inhibitor and a fibrilsdisaggregating promoter. The invented alginate oligosaccharide hasimportant values in preparing drugs for prophylaxis and treatment of ADand diabetes.

1. An alginate oligosaccharide or its derivatives represented by formula (I) or pharmaceutically-acceptable salts thereof, said oligosaccharide is composed of β-D-mannuronic acid linked by 1,4 glycosidic bonds, as shown by the following formula (I):

wherein, n represents a integer of 0 or 1 to
 19. 2. The alginate oligosaccharide or its derivatives or pharmaceutically-acceptable salts thereof according to claim 1, characterized in that the reduced terminal in position 1 of said alginate oligosaccharide derivative is carboxyl radical, as shown by the following formula (II):

wherein, n represents a integer of 0 or 1 to
 19. 3. The alginate oligosaccharide or its derivatives or pharmaceutically-acceptable salts thereof according to claim 1, characterized in that n is 2-12, preferably n is 4 to
 8. 4. A process for preparing the alginate oligosaccharide or its derivatives or pharmaceutically-acceptable salts thereof according to claim 1, which comprises the following steps: an alginate solution is reacted for about 2 to 6 h in an autoclave at pH 2˜6 and the temperature of about 100˜120° C., and adjusted pH to about 7 after the reaction is stopped.
 5. The process according to claim 4, characterized in that said alginate is sodium alginate and reacted for 4 h at the condition of pH 4 and 110° C.
 6. The process according to claim 4, characterized in that after adjusting pH to about 7, alcohol is added to precipitate; the alcohol precipitate is filtered off with suction, dehydrated, dried and desalted.
 7. The process according to claim 4, characterized in that after the alginate solution reacting for about 2 to 6 h in an autoclave at pH 2˜6 and the temperature of about 100˜120° C., an oxidant is added and reacted for 15 min to 2 h at the temperature of 100˜120° C.
 8. The process according to claim 7, characterized in that said oxidant is copper hydroxide and reacted for 30 min at the temperature of 100° C.
 9. A pharmaceutical composition containing an effective amount of the alginate oligosaccharide or its derivatives according to claim 1, and pharmaceutically-acceptable carriers.
 10. The pharmaceutical composition according to claim 9, characterized in that the composition is any one of a medicament for the prophylaxis and treatment of AD, an amyloid-β protein fibrils forming inhibitor, a medicament for the prophylaxis and treatment of diabetes, an islet amyloid protein fibrils forming inhibitor and a fibrils disaggregating promoter.
 11. Alginate oligosaccharide derivatives or their pharmaceutically-acceptable salts made by the following process: an alginate solution is reacted for about 2 to 6 h in an autoclave at pH 2˜6 and the temperature of about 100˜120° C., adjusted pH to about 7 after the reaction is stopped, and an oxidant is added and reacted for 15 min to 2 h at the temperature of 100˜120° C., wherein the alginate oligosaccharide derivatives are composed of β-D-mannuronic acid linked by 1,4 glycosidic bonds, wherein the reduced terminal in position 1 is carboxyl radical, as shown by the following formula II:

wherein, n represents 0 or an integer of 1 to
 19. 12. A method for the treatment of Alzheimer's disease in a subject, comprising: administering to the subject a therapeutically effective amount of mannuronic acid oligosaccharides represented by the following formula (I),

wherein, n represents a integer of 0 or 1 to
 19. 